Biochemical characterization of Soxhlet-extracted pulp oil of Canarium schweinfurthii Engl. fruit in Nigeria

Characterization and further development of underutilized/underexploited indigenous tropical seed oils are essential to supplement both nutritional and industrial needs of an ever-increasing African (and global) population. Before now and to our best knowledge, the previous research involved Canarium schweinfurthii Engl. fruit specific to Nigeria appear to have been more on the evaluation of seed, pulp, and essential oils (from the seed), but much less on the pulp oil. To supplement existing information, this current work has aimed to biochemically characterize the Soxhlet-extracted pulp oil of C. schweinfurthii fruit gathered from a community situated in the South-east of Nigeria. Specifically, the biochemical characterization comprised the determinations of proximate compositions, lipid peroxidation, fatty acid profile, as well as carotenoids, sterols, and tocopherols. Processing the fruit sample to pulp oil involved, among others, oven-drying, and grinding, prior to the Soxhlet extraction. Results of proximate components of C. schweinfurthii pulp oil showed the following trend: crude fat content (~ 49.32%) > carbohydrates (~ 37.93%) > moisture content (~ 8.62%) > ash content (~ 3.74%) > crude protein content (~ 0.39%) values. The lipid peroxidation attributes comprised acid (~ 23.60 mg KOH/g), peroxide (~ 33.91 mEq. O2/kg), iodine (~ 58.3 g/100 g), and saponification (~ 138.21 mg KOH/g) values. In addition to the free (~ 13.8%), saturated (~ 9.74%), and unsaturated (~ 90.26%) fatty acids, a total of fifteen (15) fatty acid methyl esters (FAMEs) spectral peaks were found, from caprylic acid (C8:0) to lignoceric acid (C24:0). Total tocopherol concentration amounted to ~ 73 mg/100 g, which comprised α, β, γ-tocopherol, and δ-tocotrienol, with fair concentrations of carotenoids and sterols. Overall, the C. schweinfurthii pulp oil—biochemically competitive with a high concentration of unsaturated fatty acid, tocopherol, and sterol, suggests strong industrial promise.

(Linzi, China). All chemicals/reagents employed in the study were of analytical grade standard.
Collection, preparation, and processing of C. schweinfurthii fruit to pulp. The freshly (mature fruit) harvested samples, obtained from various wild C. schweinfurthii trees and gathered as one batch (~ 90 g), were picked from Edem-ani community (6°51′43″N 7°20′21″E) of Nsukka local government area (LGA), Enugu State, South-east of Nigeria. Permission to collect the fruit samples was given by the farmers that owned the various plant fields, which availed the wild C. schweinfurthii trees. In addition to the collection process that adhered to the prescribed plant material guidelines, the taxonomy identification of C. schweinfurthii fruit samples was performed by Mr. Felix Okoli (plant taxonomist) at the Plant Science and Biotechnology Unit, University of Nigeria Nsukka, and voucher specimen has been deposited in the herbarium for reference purposes (Voucher reference number = PCG/UNN/0407Canarium schweinfurthii Engl [Burseraceae]). From the assembled batch, the fruit samples were randomly selected, and seeds separated following the method described by Abayeh, Abdulrazaq and Olaogun 3 with modifications, to secure the succulent fruit pulp (cotyledon). This involved washing the fruit pulp, severing it from the hard nut, slicing, and thereafter, subject to oven drying at ~ 50 °C for 8 h, before grinding using the electric blender. Subsequently, the ground C. schweinfurthii pulp was then ready for the Soxhlet extraction.
Soxhlet extraction of C. schweinfurthii pulp oil. To produce the C. schweinfurthii pulp oil, the Soxhlet extraction method using n-hexane as the solvent, previously described in the AOAC method 23 with some modifications, was employed. This involved weighed C. schweinfurthii ground fruit pulp sample (~ 15 g) with ~ 150 mL of n-hexane solvent submitted to Soxhlet extractor that operated at temperature of ~ 65 °C. The extraction period lasted for about 4 h. When the extraction had completed, the residual solvent was allowed to evaporate, and the free oil was quantified as yield, and recorded by percentage. The pulp oil was recovered, and preserved in a sample bottle, and stored at 4 °C until required for further analysis.
Analytical measurements of C. schweinfurthii pulp oil. Proximate analysis. Proximate analysis involved the determinations of crude protein, crude fat, moisture, ash, and carbohydrate contents, as well as nitrogen free extract using the AOAC method 24 with some modifications.
To determine the crude protein content, fresh oil sample (~ 0.3 g) was weighed into a Kjeldahl flask with 0.20 g catalyst. The digestion used ~ 10 mL concentrated H 2 SO 4 , 50 mL of 4% boric acid, followed by three drops of methyl red. Thereafter, 40% NaOH (25 mL) was added, after which the distillate was titrated against 0.5 N Na 2 SO 4 . With % N available, the determination of crude protein was established using the correction factor (% N × 6.25).
To determine the crude fat content, fresh oil sample (~ 0.3 g) was weighed into an extraction thimble and placed into a quick fit Soxhlet apparatus (Merck KGaA, Darmstadt, Germany) with solvent flask containing Figure 1. A schematic overview of the experimental program of this current study, showing the major stages, from procurement of C. schweinfurthii fruit samples, processing into the pulp, followed by the Soxhlet extraction of its oil, and subsequent analytical measurements. www.nature.com/scientificreports/ 25 mL of diethyl ether connected to a condenser. The extraction completed in ~ 6 h, and extract was evaporated at ~ 70 °C to remove any remaining solvent present. The apparatus was reweighed, and percentage fat was calculated as follows: To determine the moisture content, fresh oil sample (~ 0.5 g) was weighed and dried in the oven at 110 °C to a constant weight. The dish and sample were cooled and reweighed and percentage moisture content was determined and expressed as percentage.
To determine the ash content, previously weighed porcelain dishes had fresh oil sample (~ 3 g) subject to muffle furnace at 600 °C for ~ 3 h. The percentage ash content were calculated using the equation below: where: W 1 = weight of crucible; W 2 = Weight of crucible and sample; W 3 = Weight of crucible and ash.
In order to determine the nitrogen free extract (NFE), the crude fibre content had to be determined first, using fresh oil sample (~ 3 g), which had been subjected to diluted H 2 SO 4 , boiled for 30 min and filtered. Subsequently, ~ 150 mL of pre-heated KOH and drops of octanol were added, followed by boiling for ~ 30 min, and thereafter filtered. Thereafter, acetone was used to wash the sample for three times in the cold extraction unit, after which the content was dried at 130 °C for 1 h, and then ashed at 500 °C. The ash was weighed and percentage crude fibre calculated using the equation below: With this crude fibre information now available, the %NFE was determined by subtracting the sum of other fraction from 100 as follows: 100 − (% moisture + % protein + fat + fibre + ash) = % NFE. Furthermore, the total carbohydrate content was elucidated after all other components have been measured.
Lipid peroxidation analysis. Regarding the acid value, the AOAC method 24 with slight modifications was applied to the C. schweinfurtaii pulp oil. This involved the use of the mixture of ethanol and diethyl ether, 25 mL (denatured alcohol) (v/v), then 3 drops of phenolphthalein indicator neutralized with 0.1 M ethanolic KOH solution. About 0.5 mL of the oil samples were added to the neutralized solution, and finally titrated against 0.1 M ethanolic KOH solution, to reach permanent pink colour. Expressed as mg KOH/g, the acid value (A.V.) was calculated as follows: Regarding the peroxide value, the AOAC method 24 with slight modifications was applied to the C. schweinfurthii pulp oil. First, the oil (0.5 mL) was dissolved in a solvent mixture of acetic acid and chloroform (1:2). Then, KI (~ 1.3 g) was added, and mixture placed in a dark cupboard for 1 h, after which ~ 75 mL of distilled water was added, followed by 3 drops of starch indicator, and titrated against 0.05 M sodium thiosulphate. The peroxide value, expressed as millimoles of active oxygen per kilogram (mEq. O 2 /kg), was calculated as follows: S = (Vol. of Na 2 S 2 O 3 for blank -Vol. of Na 2 S 2 O 3 for sample), N = Normality of Na 2 S 2 O 3 .
Regarding the iodine value, the Wijs method as described by Firestone 25 with slight modifications was applied to the C. schweinfurthii pulp oil. The pulp oil samples (~ 0.5 g) has been mixed with chloroform (~ 5 mL) and Wijjs reagent (~ 8 mL), (which comprised ~ 9 mL of iodine trichloride and 10 g of iodine in chloroform (~ 300 mL)/ acetic (~ 700 mL) solution), swirled and placed in the dark cupboard for 1 h after which ~ 7 mL of KI and ~ 35 mL of distilled water were added, and titrated against 0.05 M Na 2 S 2 O 3 ·5H 2 O solution using starch as the indicator. A blank test was carried out simultaneously using water in place of the oil under the same conditions. Expressed as g/100 g, the iodine value (I.V.) was calculated as follows: Regarding the saponification value, the indicator method as described by Lamani et al. 20 with slight modifications was applied to the C. schweinfurthii pulp oil. The alcoholic KOH solution was refluxed, with pulp oil sample (~ 0.5 g). Thereafter, ~ 30 mL of 0.1 M of ethanolic KOH has been added, and allowed to boil for ~ 30 min under the reflux. Few drops of phenolphthalein indicator were added, followed by titration against 0.5 M HCl until the disappearance of the pink colour (end point). A similar procedure was administered to achieve the blank. Expressed as mg KOH/g, the saponification value (S.V.) was calculated as follows: After, the mixture was heated for ~ 5 min at 90 °C to achieve complete methylation process. The fatty acids methyl esters were extracted from the mixture with redistilled n-hexane, and then concentrated to ~ 1 mL for further analysis. The fatty acid methyl ester composition of the sample was analyzed by the injection of 1 μL of sample. The carrier gas was nitrogen with a split ratio of 1:20. The injector and detector temperatures were respectively 250 and 320 °C. The first ramping was at 12 °C/min, which lasted for ~ 20 min, and maintained for ~ 2 min. The second ramping was at 15 °C/min, which lasted for ~ 3 min, and maintained for ~ 8 min. The peaks of the fatty acid methyl esters were verified based on retention times with those of external standard (AccuStantard).
Determination of carotenoids, sterols and tocopherols. The method described by Czaplicki, Tańska www.nature.com/scientificreports/ The C. schweinfurthii pulp oil was successfully extracted using the Soxhlet extraction technique that employed n-hexane as the solvent and operated at 70 °C for 4 h. In particular, the oil yield of C. schweinfurthii fruit was ~ 53.69%, which somewhat resembled those data reported by Nagawa, Böhmdorfer, and Rosenau 13 , but above those data reported by Dongmo et al. 2 . Possibly, among other factors, the moisture content in the C. schweinfurthii fruit sample may have influenced the pulp oil yield of this current study. Additionally, the extent of the high oil yield may well be associated with the part of plant used. Besides, Dongmo et al. 2 understood that differences in oil yield from C. schweinfurthii fruit might depend on the place of harvest, and this is what Ndoye 28 observed when investigating C. schweinfurthii fruit resins from the East Region of Cameroon, where the Ebouete, Lomie and Mbeth species/varieties respectively recorded 8.6%, 7.6%, and 9.3% yield. Other plant seed oil yields reported lower values compared to those found in this current work, for instance, Chrysophyllum albidum varieties (oil yield range = 3.52-3.75%) 29 , Persea americana seed (oil yield = 36.93%) 30 , African star cherry (oil yield = 23.80%) 31 , seed oil of Lophira lanceolata (oil content = 40.0%) and Schoro caryabirrea (oil content = 42.0%) 32 . Therefore, the ~ 53.69% oil yield obtained for C. schweinfurthii fruit at this study makes it a promising oil resource for both industrial and nutritional purposes compared to other underutilized plants/crops.
Lipid peroxidation of C. schweinfurthii pulp oil. Among very important quality criteria in the food industry is the lipid breakdown levels of plant/seed oil. This is largely because the (lipid oxidation) process produces rancid flavours that decrease the food product's nutritional quality/safety. Also called auto-oxidation, this process can be quite complex especially across edible oils given its dependency on conditions of oxidation, and oil types 33 . The lipid and fatty acid contents of C. schweinfurthii pulp oil are shown in Table 2. Besides being of a pleasant odour with dark green colour and liquid at 28 °C, the pulp oil comprised acid (23.60 ± 2.35 mg KOH/g), iodine (58.3 ± 0.57 g/100 g), peroxide (33.91 ± 0.80 mEq. O 2 /kg), and saponification (138.21 ± 2.04 mg KOH/g) values, along with some quantities of free (13.8%), saturated (18.97%), and unsaturated (80.97%) fatty acids. Specifically, our iodine and peroxide values clearly differ from those data reported by Georges, Olivier, and Simard 4 for C. schweinfurthii pulp oil. Essentially, the acid value demonstrates, not only freshness of the pulp oil and its constituent free fatty acids, but also, the degree at which the triglycerides therein has been hydrolyzed by the lipase 29,34 . A low acid value suggests reduced degree of hydrolytic and lipolytic activities in the oil sample 32 . Of the current work, the acid value of C. schweinfurthii pulp oil (23.60 ± 2.35 mg KOH/g) appeared above those of African star apple (13.60 ± 2.35 mg KOH/g) 31 . Other workers like Agu, Ukonze, and Uchola 12 reported acid value and free fatty acid content of 0.62 mg KOH/g and 1.98% for Atilis oil, whereas Omeje, Ozioko, and Omeje 30 reported acid value and free fatty acid content of 7.86 mg KOH/g and 8.75% for P. americana seed oil, respectively.
Compared to iodine value (58.3 ± 0.57 g/100 g) of C. schweinfurthii pulp oil, there are other plant seed oils locally available within the same study area of this current work that have shown lower (iodine) values, for example, African star cherry seed oil (29.00 g/100 g) 31 and P. americana seed oil (33.21 g/100 g) 30 . A peak iodine value of an obtained oil would signal a high degree of unsaturation 35 , which if put in the context of this current study, would suggest the relatively high unsaturated fatty acids of the C. schweinfurthii pulp oil (80.97%). Notably, peroxide values serves as initial oxidation products and relatively short-lived aspects of unsaturated fatty acids 34 . Further, peroxide values increase with the levels of oxidative rancidity and decrease with the levels of antioxidants 36 . Peroxide value of C. schweinfurthii pulp oil (33.91 ± 0.80 mEq. O 2 /kg) at this current study fell below that of Avocado seed oil (~ 42.11 mEq. O 2 /kg) 30 . Essentially, the desirable quality edible oils that enhance the storage time with little-to-zero deterioration are those associated with low peroxide/high iodine values 35 . Nonetheless, the processing of C. schweinfurthii cotyledon/fruit into pulp, alongside Soxhlet extraction applied to extract the oil, may contribute to influence the lipid oxidation outcomes at this current work. This could be considered given that Abayeh 37 . Additionally, gas chromatography (GC) remains among the widely used methods in determining the fatty acid composition/profiles, particularly for animal fats and vegetable oils, together with their derivatives. For this purpose, the oils or fats are typically converted to their corresponding methyl esters 22,37 . In the modern analytical chemistry, most GC instruments operate with cross-detector analysis, incorporating the flame ionization detector (FID). Typically, besides helping in delivering a wide range of organic compounds, the FID provides a resistance to small fluctuations especially to the gas flow, and insensitive to the arising gas impurities. More so, the response of FID appears very predictable, as it adheres to the rule of equal carbon response, and capably provides a lower relative standard deviation particularly for inter-and intra-reproducibility 38 .
The GC-FID chromatogram of C. schweinfurthii pulp oil is displayed in Fig. 2. In total, there were fifteen (15) FAMEs in the external standard used, directly reflective of the observed spectral peaks, given that margaric acid methyl ester (C17:0) served as an internal standard. Importantly, the percent of saturated and unsaturated fatty acids would be estimated based on the components of the FAMEs external standard used. To further elaborate on the peaks, the fatty acid profile of C. schweinfurthii pulp oil, based on compounds, retention time, concentration, carbon chain ratio, and chemical formulae is shown in Table 3. The fatty acid profile of C. schweinfurthii pulp oil, arranged in the ascending order based on their respective chemical formulae and retention times. For emphasis, the caprylic acid obtained the least carbon chain ratio (C8:0), whereas the lignoceric acid obtained the highest carbon chain ratio (C24:0). Moreover, the oleic acid (C18:0) obtained the highest fatty acid concentration of 74.56%, whereas the caprylic acid (C24:0), capric acid (C10:0), lauric acid (C12:0), and myristic acid (C14:0) were almost not detected in the oil. Georges, Olivier, and Simard 4 reported high content of oleic acid (89.4%) and stearic acid (67.7-84%) of C. schweinfurthii pulp oil from the respective liquid and semi-solid parts of the oil, both well above those reported in this current study (oleic acid = 74.56%; stearic acid = 8.57%). The percentage oleic acid found in this study is related to total concentrations of the oleic acid (C18:1 n-9) and its isoforms (potentially C18:1 n−7 and C18:1 n−12, vaccenic acid and petroselinic acid, respectively). Besides the oleic acid being considered the topmost monounsaturated fatty acid in the human diet, the consumption of such (monounsaturated) fatty acid would help to increase the high-density lipoprotein (HDL), and decrease the low-density lipoprotein (LDL) cholesterol types 39,40 .
The generalized fatty acid content values of C. schweinfurthii pulp oil, previously reported by Maduelosi and Angaye 8 , included oleic (~ 36%), linoleic (~ 28%), palmitic (~ 26%), and stearic (~ 7%) acids, all of which appears not to be in agreement with those of this current study. A number of reasons could be responsible for www.nature.com/scientificreports/ such differences in fatty acid content values at this current study, for instance, the location of the cultivated C. schweinfurthii tree crop, harvest time, as well as the applied oil extraction method(s). Nonetheless, there are other reported fatty acid profiles of resembling extracted seed oils that can be compared with those of this current work. For example, the oleic acid content of C. albidum seed oil (30.21%) 31 and of P. americana seed oil (40.33%) 30 fell below that of C. schweinfurthii pulp oil at this current study. Essentially, the presence of fatty acids could provide several physiological benefits to the human immune system 41 , which are crucial for body metabolism/energy production 42 . Given its high oleic acid concentration, the C. schweinfurthii pulp oil could help in managing the human dietary cholesterol by keeping the blood's Low Density Lipoprotein (LDL) in check 43 .
Moreover, there are a number of factors that can influence the degree by which the auto-oxidation process takes place, which can include fatty acid composition, light, metal ions, polyphenols, temperature, and tocopherols 34 . The concentration of campesterol of C. schweinfurthii pulp oil (31.313 µg/100 mL) at this current study fell below those of cold pressed coconut oil 48 , and together with sitosterol, competes well with other edible oils reported elsewhere 49,50 . This current result of C. schweinfurthii pulp oil appears to relate with another previous   51 . Particularly, the presence of campesterol and sitosterol in C. schweinfurthii pulp oil of this current work was actualized, owing to the Soxhlet extraction that employed organic solvent with a moderate temperature 22 . As a plant sterol, campesterol possesses the anticarcinogenic capacity, which could lower the cholesterol 52 . Considering the apparent synergistic stimulatory effect of sitosterol on the immune system, it would be desirable to consume sufficient (sitosterolcontaining) unprocessed/unrefined plant foods 53 .
Generally, plant as well as vegetable oils comprise a number of bioactive constituents, which include tocolrelated compounds, e.g., tocopherols, tocotrienols, etc. Specifically, tocopherols are well known vitamin E compounds that possess saturated phytyl chain, whereby the α-, β-, γ-, δ-types are differentiated based on the location and number of methyl constituents within the chroma ring 54 . As demonstrated in Table 4, the total concentration of tocopherols in C. schweinfurthii pulp oil was ~ 73 mg/100 g, which comprised α-, β-, and γ-tocopherol, as well as δ-tocotrienol ,detected at varying concentrations, which appeared above those that Franke et al. 45 reported for rapeseed oil (~ 68.0 mg/100 g). Further, the relative abundance of tocopherol in C. schweinfurthii pulp oil makes it a reliable source of natural antioxidant. Moreover, previously reported vegetable oils of corn and soybean seed 45,55 , as well as palm oil 56 signals that variations in the amounts/concentrations of carotenoids and tocopherol should be expected in oil seeds. Additionally, the carotenoid content of the oils of plant seed would supplement their antioxidant potential/value 55 . A high concentration of α-and γ-tocopherol has been reported in canola, sunflower and corn oil 57 . The tocopherol, although needful in tiny concentrations to maintain good human health 45 , which being present in the C. schweinfurthii pulp oil of this current study would suggest it as promising in scavenging the free radicals 20 . Besides, processing methods would contribute to considerably reduce the quantities of carotenoids usually detected in the raw nature of plant oil 56,58 .

Conclusions
The proximate, lipid oxidation, fatty acid profile, carotenoids, sterols, and tocopherols of Soxhlet-extracted pulp oil of C. schweinfurthii fruit specific to South-east of Nigeria has been successfully investigated. For emphasis, the processing of the fruit sample to pulp oil involved, among others, oven-drying, and grinding, prior to the Soxhlet extraction, the latter of which employed n-hexane as the solvent, and resulted in promising pulp oil yield. Further, the proximate components, lipid peroxidation and fatty acid features/profile, together with the concentrations of sterols, tocopherols and carotenoids have cumulatively helped to demonstrate the biochemical importance of the C. schweinfurthii pulp oil. Potentially, the instances of high quantities of oleic acid, carotene, as well as tocopherol makes the pulp oil of this study nutritionally important, and biochemically competitive with strong industrial promise. Given the findings of the current work, it would be useful for future studies to investigate the group of flavonoids, which includes anthocyanins and condensed tannins, given that the content of these two groups could help to further unravel the richness of the pulp oil. In addition, future studies could also investigate the changes in lipid peroxidation of the C. schweinfurthii pulp oil when subject to varied storage conditions, because such new data would provide additional relevant information, not only about its biochemical/nutritional status but also its industrial potential.